PROJECT SUMMARY/ABSTRACT The overarching goal of this application is to identify novel mechanisms by which enteroviruses infect the human intestinal epithelium. The events associated with enterovirus infections of the human intestinal epithelium remain largely unknown, largely due to the lack of suitable in vivo models that recapitulate the enteral route of infection in an immunocompetent setting and the inability of standard cultured cells to recapitulate the multicellular nature of the GI epithelium. Using two parallel three-dimensional (3-D) cell models of the human intestinal epithelium recently developed in our laboratory, including a primary stem cell-based model, we have identified several unique mechanisms used by enteroviruses to infect the GI epithelium. The studies proposed in this application will provide important insights into (1) the role of non-lytic release in the enterovirus life cycle in the human intestinal epithelium, (2) the cell-type specific nature of enteroviral infections in the GI tract, and (3) the role of epithelial host innate immune signaling in the detection of enteroviral infections. These goals are premised on the central hypothesis that intestinal cell-associated pathways directly impact enterovirus pathogenesis in the human GI tract. Our proposal pioneers research into a variety of aspects of the molecular mechanisms of enterovirus-GI cell interactions. Notably, our research will also illuminate virus specific-pathogenic pathways, which may explain why some enteroviruses are relatively well-tolerated, and others cause severe disease. Given our extensive expertise in enterovirus research, specifically studies related to the GI tract, we are uniquely positioned to perform these studies, which will provide new paradigms for our understanding of enterovirus infections of the GI epithelium.